Do sheep worms occur in

wild hares and rabbits in Australia?

Marina Hsiang Hua Tai

B.Vet.Sc. (The Federal University of Goiás, Brazil)

Supervisors:

Dr Philip Stott

Dr Ryan O’ Handley

Submitted for the Degree of Master of Science

In the School of

Animal and Veterinary Sciences,

Faculty of Sciences,

University of Adelaide

May 2013

Declaration

This work is the result of my own investigation. The content herein has not been accepted for the a ward of a ny ot her degree or di ploma i n any university or o ther te rtiary in stitution to

Marina T ai a nd, t o t he be st of m y know ledge and be lief, c ontains no m aterial pr eviously written or published by any other persons except where due reference is made in the text.

I give m y c onsent f or t his t hesis w hen de posited i n t he U niversity Library, be ing m ade available for loan or photocopying, subject to the provisions of the Copyright Act 1968.

I also give permission for the digital version of my thesis to be made available on the web, via the U niversity’s d igital r esearch r epository, th e Library catalogue, th e A ustralian D igital

Thesis P rogram ( ADTP) a nd a lso t hrough w eb s earch engines, unl ess pe rmission ha s be en granted by the University to restrict access for a period of time.

September 2013

ii

Acknowledgements

Working on t his master´s project has been an unforgettable pleasant experience and I would not have been able to get through this awesome adventure without the support and help from the following people:

Firstly, I would like to thank my supervisors Dr Philip Stott and Dr Ryan O’ Handley for their inspiring guidance, constant support and for giving me the opportunity to see Australia with an exciting adventurous perspective through fieldwork. I truly appreciated working with you!

I would like to thank Dr David J. Jenkins who I have had the pleasure to work with in New South Wales and kindly sheltered me in his house during the period of sample collection in the area. I also thank Dr Miroslaw Stankiewicz for his help with spicule morphology. It was so nice to have met you.

Thank you to Dr Farhid Hemmatzadeh and Dr Behzad Khansarinejad for their friendship and helping me out in the lab, teaching me techniques and principles of molecular assays which I will continue to use throughout my research career.

I am very grateful to the members of the Conservation and Wildlife Management branch of the S porting S hooters A ssociation o f A ustralia, pa rticularly P atrick K erin an d A lex McDonald, V ince R ujero a nd J ohn S chulze w ho pr ovided m e t he s amples f or m y project. Special t hanks also go t o J ohn M atthews from the Department of P rimary Industries, (DPI Hamilton, Victoria).

I must also acknowledge friends and colleagues who were there for me every day, giving me intuitive advice, source of warmth and plenty of fun distractions. Thank you Tai Yuan Chen, Rebecca Athorn, Richard Bosworth, Lesley Menzel, Ruidong Xiang, Vaibhav Gole and Ali Javadmanesh.

Finally, and very importantly, I would like to thank my family, especially my mom and dad, who have assisted me throughout the course of my master’s work. Loving thanks must go to my boyfriend Faisal Quadri for his enduring patience and support, understanding and love. iii

Abstract

Areas of common grazing between hares (Lepus europaeus), rabbits (Oryctolagus cuniculus) and sheep ( Ovis aries) are widespread i n s outh e astern A ustralia. For much of t he year, lagomorphs are exposed to the infective larvae of the nematode parasites of livestock on farm pastures. Given that gastrointestinal parasites are a major problem for sheep graziers and that in experimental circumstances sheep helminths are able to develop in rabbits and hares, free- living l agomorphs w ere i nvestigated r egarding carriage of ovi ne n ematode pa rasites u nder field conditions. 110 hares and 88 rabbits were shot by hunters in paddocks previously grazed by s heep or i n vi neyards ne ar s heep pa stures. Lagomorphs w ere acquired f rom N ovember 2010 t o A ugust 2012 f rom t he A delaide r egion of S outh A ustralia, t he w estern di strict o f Victoria and central western New S outh W ales. Total helminth counts and examinations of spicule m orphology w ere pe rformed. P CR w as u tilized t o c onfirm f indings. M y study revealed t hat t he r uminant w orm, Trichostrongylus colubriformis, is c ommon i n ha res (prevalence 3 2.7%) a nd a lso, oc casionally, o ccurs i n r abbits ( prevalence 3.4 %). S tatistical analysis showed no significant effects of age or sex of either hares or rabbits, in prevalence of worms ( P > 0.05). C hi-Square and F isher E xact t ests w ere p erformed an d s howed t hat, i n general, nematode parasite infestations were not significantly different in hares or rabbits (P > 0.05) f or a ll r egions e xamined. H owever, w hile t he r uminant ne matode T. colubriformis occurred more frequently in hares, rabbits were more commonly infected with the lagomorph- specific Trichostrongylus retortaeformis (prevalence 61.4 %). The l agomorph worm Graphidium strigosum was mainly found in rabbit stomachs obtained from New South Wales. The ruminant nematode Trichostrongylus rugatus, was identified infecting four hares and one rabbit f rom th e A delaide r egion, S outh A ustralia, a nd is r eported f or th e f irst time in w ild lagomorphs. Cross-transmission of nematodes between lagomorphs and sheep in the natural environment is much more prevalent than previously believed. Further studies will contribute important information to assist sheep producers manage nematode gastrointestinal parasites and m ay a lso l ead t o ne wly i dentified c auses f or t he de clines of l agomorph popul ations i n various parts of the world.

iv

List of Figures

Chapter 1

Figure 1.1 ------16

Figure 1.2 ------16

Figure 1.3 ------17

Figure 1.4 ------18

Chapter 2

Figure 2.1 ------22

Figure 2.2------23

Figure 2.3 ------24

Figure 2.4 ------25

Figure 2.5 ------26

Figure 2.6 ------27

Figure 2.7 ------28

Figure 2.8 ------30

Figure 2.9 ------32

Chapter 3

Figure 3.1 ------37

Chapter 4

Figure 4.1 ------44

Figure 4.2 ------44

Figure 4.3 ------45

Figure 4.4 ------48

Chapter 5

Figure 5.1 ------53

v

List of Tables

Chapter 2

Table 2.1 ------31

Chapter 3

Table 3.1 ------38

Chapter 4

Table 4.1 ------40

Table 4.2 ------41

Table 4.3 ------42

Table 4.4 ------45

Table 4.5 ------46

vi

Summary

Chapter 1 Literature Review ...... 9 1.1 Wild lagomorphs in Australia ...... 9 1.2 Lagomorph parasitic nematodes ...... 9 1.3 Ovine parasitic nematodes ...... 10 1.4 Pathogenicity ...... 10 1.5 Immunity ...... 11 1.6 Anthelmintic resistance ...... 13 1.7 Quarantine ...... 13 1.8 Host specificity ...... 14 1.9 Evolution ...... 15 1.10 Distribution of lagomorphs and sheep in Australia ...... 15 1.11 Life cycle and nematode availability on pasture ...... 18 Chapter 2 ...... 21 Morphology ...... 21 2.1 Introduction ...... 21 2.2 Species descriptions ...... 21 2.2.1 Graphidium strigosum (Dujardin, 1845) Railliet and Henry, 1909 ...... 21 2.2.2 Trichostrongylus retortaeformis (Zeder, 1800) ...... 23 2.2.3 Trichostrongylus colubriformis (Giles, 1892) ...... 24 2.2.4 Trichostrongylus rugatus (Mönnig, 1925) ...... 25 2.2.5 Passalurus ambiguus (Rudolphi, 1819)...... 26 2.3 Methods ...... 27 2.3.1 Study sites ...... 27 2.3.2 Animal sampling ...... 32 2.3.3 Nematode sampling ...... 33 Chapter 3 ...... 34 Molecular assesment: Polymerase chain reactions ...... 34 3.1 Nematode diagnostics ...... 34 3.2 Material and methods ...... 35 3.2.1 DNA extraction ...... 35 3.2.2 Primer design ...... 35 3.2.3 Conventional PCR set up ...... 36 Chapter 4 ...... 40 Results ...... 40 4.1 Morphological and molecular diagnosis ...... 40 vii 4.1.1 Prevalence ...... 40 4.1.2 Necroscopic examination ...... 48

Chapter 5 ...... 49 Discussion ...... 49 5.1 Prevalence ...... 49 5.2 Nematode species distribution ...... 50 5.3 Evolution ...... 52 5.4 Clinical disease ...... 52 5.5 Spicule morphology ...... 53 5.6 Morphometry...... 54 5.7 Molecular assay ...... 54 5.8 Permissiveness and anthelmintic resistance ...... 55 5.9 Future directions ...... 56 References ...... 57

viii Chapter 1

Literature Review

1.1 Wild Lagomorphs in Australia

Two wild lagomorph species occur in Australia, the European rabbit (Oryctolagus cuniculus Linnaeus, 1758) and t he E uropean br own ha re ( Lepus europaeus Pallas, 1778) ( Class – Mammalia, O rder – Lagomorpha, F amily – Leporidae, F ischer, 1817) . B oth s pecies were introduced ca. 1860 by British settlers (Rolls, 1984). The rabbit represents a major pest in the continent and causes serious environmental and agricultural damage (Myers, 1986; Williams et al., 1995; Jarman and Stott, 2008). On the other hand, the hare, which is currently not at plague d ensities, is r arely subject to c ontrol (Stott, 2003a ; Page e t a l., 2008 ). T hese w ild herbivores h ave co -existed w ith l ivestock i n pa stures a nd ope n s hrublands f or m any years. However, some important interactions between them have not yet been investigated.

1.2 Lagomorph parasitic nematodes

The helminth nematode fauna that naturally occurs in lagomorphs is diverse. The nematodes Graphidium strigosum (Allgoewer, 1997), Trichostrongylus retortaeformis (Dunsmore, 1966; Boag, 1987a; Tenhu, 1998), and Passalurus ambiguus (Boag and Iason, 1986; Bordes et al., 2007; Beck and Pantchev, 2009) have been the most frequently recorded nematode species in free-living hares and rabbits, worldwide and in Australia. However, presumably because of a founder effect, a number of nematode parasites that occur in European hares and/or rabbits elsewhere on Earth do not occur in Australia, such as Obeliscoides cuniculi, Trichuris leporis, Strongyloides sp., Protostrongylus spp., Nematodiroides zembrae and Dirofilaria scapiceps (Evans, 1939 ; Bull, 1953 ; Mykytowycz, 1956 ; Hesterman a nd K ogon, 1963 ; Soveri a nd Valtonen, 1983 ; Boag, 1985 , 1987a; Murray et a l., 1997 ; Tenhu, 1998 ; Allan e t a l., 1999 ; Foronda et al., 2003; Newey et al., 2005; Bordes et al., 2007; Stojanov et al., 2008; Dubinsky et al., 2010; Tizzani et al., 2011; Lukešová et al., 2012).

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1.3 Ovine parasitic nematodes

The sheep and wool industry contribute significantly to the Australian economy but ruminant gastro-intestinal nematode parasites (also kno wn a s “ round w orms”) cause s ubstantial economic losses estimated at $369 m illion per annum, making them the major sheep health concern (Holmes et al., 2006). The most important helminths which occur in sheep belong to the super-family Trichostrongyloidea (Lichtenfels e t a l., 1997 ) which occur i n the g astro- intestinal tr act (Durette-Desset, 1992 ; Chilton e t a l., 2006 ). The f ollowing a re s ome of t he most c ommonly f ound species of n ematodes i n s heep a nd r epresent a constraint t o s mall ruminant pr oducers w orldwide: Haemonchus contortus, a lso know n as t he b arber’s p ole worm; Teladorsagia (Ostertagia) circumcincta or b rown s tomach w orm; a nd Trichostrongylus spp. or black scour worms. The first two species and Trichostrongylus axei are found in the abomasum (true stomach) of the host, other Trichostrongylus spp. occur in the small intestine (Cole, 1980) where Nematodirus spathiger, N. filicolis, N. abnormalis and N. helvetianus - thin-necked i ntestinal worms - are found (Beveridge and Ford, 1982), a nd whipworms such as Trichuris ovis, T. globulosa and T. skrjabini together w ith Chabertia ovina and Oesophagostomum venulosum are found in the large intestine (Pullman et al., 1988; Hutchinson, 2009). These major livestock parasites not only decrease weight gain and reduce quality of wool in sheep, but also cause significant animal mortalities and incur significant costs with treatment (McLeod, 1995).

1.4 Pathogenicity

Ovine trichostrongylosis is associated with a number of symptoms such as inappetence - food intake m ay b e r educed by 2 0% (Holmes, 1985 ), a naemia a nd, v ery i mportantly, di arrhoea. Diarrhoea i s a m ajor pr oblem f or s heep pr oducers be cause, be sides t he w eight l oss, t he animals’ wool gets soiled around the breech and this decreases its value and also makes them susceptible to m yiasis ( or “ blowfly s trikes”) w hich c an p roduce f atalities if le ft u ntreated (Heath a nd B ishop, 198 6; Broughan a nd W all, 2007; Jacobson e t a l., 2 009; Williams a nd Palmer, 2012). According to Williams and Palmer (2012), sheep do not necessarily need to be heavily i nfested w ith w orms t o p resent d iarrhoea. T his can also o ccur as a r esult o f t he immune-pathological processes of the sheep itself, especially in animals of post-weaning age, which ar e m ainly affected b y t he gastro-intestinal p arasites Teladorsagia circumcincta and other members of Trichostrongylina. Large worm burdens of Trichostrongylus spp. can cause critical enteritis, severe villus atrophy, impaired villus/crypt ratios and hyperplasia of goblet cells (Pullman et al., 1991) as well as thickening of the mucosa. Neutrophils and lymphocytes 10 can also be found infiltrating the affected area (Holmes, 1985). Steel et al. (1980) estimated that 950-3000 larvae of T. colubriformis per week produced impairment in wool and growth in s heep. A n e stimation of ove r 1000 T. calcaratus produced fatal in festations in r abbits (Sarles, 1934) and ca. double this amount of worms are necessary to induce clinical disease in a one -year-old s heep (Soulsby, 1968 ). M oreover, i t ha s be en s uggested t hat, in r abbits, T. retortaeformis affects l itter s urvival as p arasitized f emales h ave d ecreased m ilk p roduction and consequently, smaller kittens with reduced chances of survival (Dunsmore, 1981). Body weights of rabbits a nd snowshoe ha res Lepus americanus have also b een i mpacted b y T. retortaeformis, presumably due to villar atrophy and reduced absorptive capacity in the small intestine (Barker and Ford, 1975; Jacobson et al., 1978 ). Iason and Boag (1988), how ever, determined t hat n either body w eight o r r eproductive p erformances w ere af fected b y h igh infestations of this nematode when parasitizing mountain hares Lepus timidus. On the other hand, e xperiments c onducted w ith f emale mountain ha res s howed t hat t reating T. retortaeformis infections increased fecundity (Newey and Thirgood, 2004).

1.5 Immunity

Generally, he lminth pa rasites i nduce a Type 2 i mmune r esponse i n t he hos t, w hich i s characterized b y b oth T he lper t ype 2 lymphocytes (with pr oduction of s ome s pecific interleukins, followed by a large production of eosinophils and goblet cells hyperplasia); and later, a m ore e ffective adaptive r esponse w hich, t hrough v arious m echanisms ( i.e. c lass switched immu ne-globulins a nd a ctivated le ukocytes), w ill le ad to th e d islodgement o f th e parasites a nd t heir consequent e limination (Maizels et al ., 2 012). Immune r esponse a gainst parasitic n ematodes can ei ther b e d isplayed a gainst s pecific s tages o f the p arasite o r b e directed to both larval and adult phases (Balic et al., 2000; Hein et al., 2010).

In sheep, antibodies IgE, IgG1 and IgA rise systemically and locally in the intestines with a fast proliferation of eosinophils and mast cells (Balic et al., 2000). The rise of activated mast cells associated with low worm burdens in sheep (Hein et al., 2010) might help explain how the inflammatory mechanism prevents infective larvae from establishing in the host mucosa (Meeusen, 1999).

Experimental Cooperia oncophora infections in c attle s howed th at the host’s i mmune regulatory s ystem s uffers a lterations d uring p arasite in fections a nd r esponses m ay va ry according to host species as well as to the type of nematode involved (Li and Gasbarre, 2009). Economic l osses a re a lso due t o hos t´s i mmune r esponse i n t he pr esence of s ubclinical infections with gastro-intestinal nematodes. There is a re-prioritization of nutrient utilization 11 and diversion is towards activation of the immune process to the detriment of regular growth and production (Page et al., 2008).

Along w ith vi ruses and b acteria, s trongylid n ematodes h ave d eveloped s trategies t o a void being t argeted b y host antibodies. A s tudy ha s d emonstrated t hat t he i nfective l arvae of T. colubriformis may evade host’s immune response by presenting antigenic variation. Maass et al. (2009) identified at least three different epitopic forms in this species. Another nematode parasite s trategy is th e s ecretion o f immu ne-modulators i .e. cysteine pr otease i nhibitors or cystatins. T hese pr oducts s uppress T cel l responses t o n ematode i nfections and, t herefore, modulate host immune responses (Hartmann and Lucius, 2003).

Generally, a host age-dependence is observed and immune-competence is assumed to be built up g radually w ith c ontinual e xposure t o i nfective l arvae e.g. T. retortaeformis in r abbits (Cornell et al., 2008) and T. colubriformis in sheep (Dobson et al., 1990). This is dependant not on a ny particular age but on t he force of infection reaching a peak more rapidly – at a younger age – when it is high, or reaching it more slowly – in older animals – when it is low. This pattern called the “peak shift” is very well described by Woolhouse (1998) and Cattadori et al . (2005). In a n e xperimental in fection w ith o vine T. colubriformis, M usogong e t al. (2004), for example, showed that m ean n ematode and faecal egg counts in juvenile rabbits were s ubstantially hi gher t han i n a dult r abbits. T he s ame pa ttern w as f ound i n a s tudy conducted by C hylinski e t a l. (2009) using w ild r abbits a nd na turally acquired T. retortaeformis. T he authors s howed, i n a ddition, t hat ne matode l ength d ecreased i n ol der rabbits, suggesting that host’s acquired immunity may influence nematode growth as well as nematode egg production. However, G. strigosum seemed to modulate immunity in rabbits as no decrease in worm numbers of this species could be observed in the hosts (Murphy et al., 2011). Furthermore, the ability of host modulation of G. strigosum may positively influence infections by T. retortaeformis (Lello et al., 2004).

In addition t o a ne gative c orrelation be tween host a ge and pa rasite a bundance there i s, apparently, evidence that a component of “season of birth” might influence development of the host immune response in wild rabbit populations, naturally infected by T. retortaeformis (Cornell e t a l., 2008 ). Rabbits bor n i n e arly s pring, f or e xample, ha d a s tronger i mmune response a s oppos ed t o one s bor n in a utumn, w hen i mmune a bilities w ere c onsidered t he poorest (Cornell et al., 2008). In any case, immunity is decreased during host pregnancy and

12 lactation (Khan and Collins, 2004; Cattadori et al., 2005), co-infection with the myxoma virus (Cattadori et al., 2007), poor nutrition and exposure to severe stress (Sedlak et al., 2000).

1.6 Anthelmintic resistance

Sheep producers rely mainly on t he routine use of broad-spectrum chemical anthelmintics to control parasitic diseases but the development of anthelmintic resistance has been reported for the majority of the anthelmintic drugs (Coles et al., 2006; Papadopoulos, 2008; Torres-Acosta et al., 2012). Despite distinct modes of action, resistant strains of parasites began to appear a few years after the introduction of each anthelmintic group, in particular within Haemonchus contortus, Teladorsagia (Ostertagia) circumcinta and Trichostrongylus spp. (Sangster an d Dobson, 2002 ; Kaplan, 2004 ). A ccording t o P apadopoulos ( 2008) t he mechanism f or t he development of a nthelmintic r esistance i s t he s urvival of a s mall num ber of w orms dur ing drench t reatments, contaminating t he pa sture with r esistant l arvae i n t he s ubsequent generation. There is also a percentage contribution to the population by worms in refugia, i.e. ones not e xposed t o t he dr enches, but t here i s a s election pr essure against s ubsequent generations of susceptible parasites if the same class of anthelmintic is reused, leading to a dominance of resistant parasites. Frequency of treatments and sub-therapeutic doses are other factors which contribute to the perpetuation of resistant heterozygous worms therefore, there is a s election of resistant lineages of parasites (Egerton et al., 1988). Goats can also develop resistant strains of parasites that may be passed to sheep, especially in common grazing areas (Jackson, 1993).

1.7 Quarantine

Quarantine at t he pr operty l evel constitutes a n aspect o f great imp ortance in th e s trategic control process for coping with worms. Sheep being introduced onto a farm may be retained in sheep yards, treated with an anthelmintic, and held until viable propagules are eliminated, before t he i ntroduced s heep a re r eleased i nto g razing pa ddocks t o j oin t he e xisting f lock (Swarnkar and Singh, 2012). Under feasible conditions, a minimum 24-36 h waiting period and treatment with an anthelmintic drug like monopantel, for example, before release into the flock o f n ewly acquired s heep i s r easonable, a ccording t o Sager et al . (2010). Combined anthelmintic therapy with quarantine is an important tactic against resistance. Some graziers never quarantine and treat the animals brought on to their farms whilst others do i t properly (Coles, 1997; Morgan et al., 2012).

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1.8 Host specificity

Wild animals are able to cross barriers established to protect livestock and play a role in the cross-transmission o f s ome p arasitic n ematodes a lso c ommon to d omestic r uminants. I n Sweden, for example, free-ranging deer of several species were recognized to have a potential to br each f arm bounda ries a nd s hare s everal he lminth s pecies w ith s heep (Nilsson, 1971 ; Borgsteede, 1982 ). S heep a re a lso know n t o s hare ne matode pa rasites w ith goats (Torres- Acosta and Hoste, 2008), cattle, camels (Kumsa and Wossene, 2006), horses (Bucknell et al., 1995), pigs (Roberts, 1940), possums (Stankiewicz et al., 1996) and reindeer (Hrabok et al., 2006). Occasionally and incidentally, ovine nematodes have been found even in monkeys and humans (Joe, 1947 ; Lattès e t a l., 2011 ). Five t o 15 a dult ne matodes of t he genera Oesophagostomum sp, Trichuris sp, and the r uminant ne matode Haemonchus sp. w ere retrieved from p lateau p ikas ( Ochotona curzoniae) i n C hina (Wang e t a l., 2009 ). A s mall number of wild h ares and r abbits ha ve be en f ound pr esenting na tural i nfections of s heep worms (Boag, 1987b; Saulai and Cabaret, 1998; Beck and Pantchev, 2009; Usai et al., 2012). Surveys reported num bers of ovi ne Teladorsagia circumcincta, Nematodirus spp., Trichostrongylus axei, T. colubriformis and T. vitrinus (Mykytowycz, 1956; Mackerras, 1958; Hesterman a nd Kogon, 1963 ; Boag, 1972 , 1987a; Saulai a nd C abaret, 1998 ). The ovi ne nematodes T. colubriformis and Strongyloides papillosus have been successfully established in laboratory rabbits e.g. (Sommerville, 1963; Nwaorgu and Connan, 1980; Hoste et al., 1988) and S tott et al. (2009) showed t hat t he E uropean br own ha re i s a lso permissive to Teladorsagia circumcincta and t hat Trichostrongylus colubriformis, T. rugatus, T. vitrinus and small numbers of Nematodirus spp. and Cooperia sp. have the ability to develop to the adult stage in European hares. However, the hares studied by Stott et al. (2009) were captive juvenile ha res i n a hi ghly a rtificial e nvironment and co nsequently m ay h ave experienced stress-induced immunosuppression, and hence the study may not represent the situation in the field.

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1.9 Evolution

The sub-order Trichostrongylina is a large and well-distributed taxon of nematode parasites which were grouped together mainly by their morphological and evolutionary traits (Durette- Desset, 1985 ). T he t hree s uper-families w ithin th is s ub-order: T richostrongyloidea, Molineoidea and Heligmosomoidea (Durette-Desset and C habaud, 199 3), present nematode parasites of l agomorphs t hat oc cur i n t he s ame s ub-family ( or genus i n t he c ase o f Trichostrongylus) a s th ose w hich p arasitize r uminants. Graphidium strigosum, know n t o parasitize lagomorphs and Haemonchus contortus, which affects sheep (and, to a lesser extent cattle), b oth in fect th e g astric mu cosa o f th eir h osts. P hylogenetic s tudies b ased in morphological characters a nd m olecular analyses ha ve not yet e lucidated, i n e volutionary terms, t he s pecific r elationship be tween t hese t wo s pecies, how ever, i t a ppears t hat t hey probably had a common ancestor within the Trichostrongyloidea (Chilton et al., 2001; Gouÿ de Bellocq e t a l., 2001 ; Audebert e t a l., 2005 ). In t erms of e volution, t he m ost pr obable hypothesis is that adaptation of trichostrongyloids to lagomorphs would have occurred prior to adaptation to ruminants and that the species of parasites existent in the latter, originated from those of lagomorphs (Durette-Desset et al., 1999; Audebert and Durette-Desset, 2007). The s imilar he rbivorous ha bits of L agomorpha a nd R uminantia w ould ha ve e xposed t he evolving ruminant species to the helminths of the lagomorphs, providing the degree of contact necessary for hos t-switching t o be come pos sible (Chabaud, 1965 ; Audebert a nd D urette- Desset, 2007).

1.10 Distribution of lagomorphs and sheep in Australia

The distribution of the hare is over an area of south-eastern Australia occupying c. 700,000 km2 (Fig. 1.1) (Jarman, 1986; Stott, 2003b) which is largely within Australia´s Mediterranean climate zone, characterized by mild humid winters and hot, dry summers. The land is mainly used for agriculture, constituted by integrated wheat and sheep grazing through much of the year on pastures and, in the summer, on crop stubbles (Stott and Harris, 2006). In A ustralia, t he r abbit occupies an a rea o f 4,5 00,000 km 2 (Fig. 1.2) i ncluding almost t he entire distribution of both the hare and the sheep (Fig. 1.3), and hence there are high levels of overlap between the distributions of the three species. In many areas, the overlap also occurs on a very fine scale, and the home ranges of hares (up to 200 ha) include areas also used by rabbits and sheep, including flocks on neighbouring farms (Stott, 2003b).

15

Fig.1.1 Distribution of the hare in Australia (Jarman and Stott, 2008)

Fig. 1.2 Distribution of the rabbit in Australia (Jarman and Stott, 2008)

16

Fig.1.3. Distribution of sheep and lambs in Australia (Australian Bureau of Statistics, 2006)

17 1.11 Life cycle and nematode availability on pasture

The Trichostrongyloidea has a direct life cycle with a f ree-living stage and a parasitic stage. The duration o f t he fre e-living stage is fro m 3 -9 da ys. If not equivalent, t he larval and t he maturation pe riods, a s well t he pre-patent pe riod, are relatively shorter in l agomorph hosts than i n r uminant hos ts (Audebert a nd Durette-Desset, 2007 ). T he l ife c ycle o f a trichostrongylid is illustrated below (Figure 1.4).

L3 L4

Free-living phase Parasitic phase L2 adult (on pasture) (in host)

L1 egg

Fig. 1.4 General life cycle of a trichostrongylid nematode parasite. L1 to L4 are larval stages.

The epidemiology of the helminth parasites of sheep has been subject of many studies. In the winter rainfall areas there is a d ominance of T. circumcincta and other Trichostrongylus spp. These nematodes are not as sensitive to desiccation as H. contortus, a worm that is prevalent in s ummer r ainfall a reas (O'Connor e t al., 200 6). Despite evidence s uggesting t hat o ne Trichostrongylus spp. or another may be more prevalent in sheep in some regions rather than others (Gray and Kennedy, 1981; Beveridge and Ford, 1982), the Trichostrongylus spp. that commonly occur in sheep in Australia are four: T. colubriformis, T. vitrinus, T. rugatus and T. axei (Pullman et al., 1988; Beveridge et al., 1989a; Bailey et al., 2009).

Influence of th e c limate o f a p articular r egion is a n imp ortant f actor on t he s urvival and development of the free living stages and hence a factor influencing the prevalence of each

18 species. Hot dry summer months are unsuitable for larval development; however, numbers of T. circumcincta and Trichostrongylus spp. may survive in faecal pellets (Young, 1983) from which they migrate onto pasture following onset of slight falls of rain. Larvae which survive in faeces over summer may lead to serious infections in the following autumn (Southcott et al., 1976). However, without rain, the emergence of infective larvae is considerably reduced. Pastures on f arms c an be i nfested f or m uch of t he year w ith l arge nu mbers of l ivestock infective larvae. Agneessens (1997) and Tessier and Dorchies (1997) have recorded densities of larvae of ruminant origin exceeding 1400 kg-1 dry matter (DM) and hares eat c. 190 g dry matter day-1 (Stott, 2008). Considering typical levels of infestation of sheep and their grazing hence defaecation patterns, hares are much more likely to be exposed to infective larvae of ovine or igin t han of l agomorph or igin; a nd c onsidering t he pr oportions of ovi ne pa rasitic nematodes that are resistant to the various anthelmintics, it is inevitable that hares would be ingesting th e l arvae o f resistant s trains o f n ematodes. H owever, t he f ate o f t hose l arvae i s unknown.

19

Conclusion There is a h igh level of spatial overlap between sheep and hares in Australia (Stott, 2003b, 2008) and experimental infections with ruminant nematodes demonstrated susceptibility and even permissiveness of lagomorph hosts to internal parasites of sheep (Hoste and Fort, 1992; Audebert e t a l., 2003 ; Stott e t a l., 2009 ). H owever, i t remained to be ascertained if sheep nematodes could be found, in significant numbers, in hares and in rabbits in the field under natural conditions. If this occurred, it would, perhaps, have implications for the management of t he s pread of resistant w orms in liv estock and i mpact hare a nd rabbit popul ations themselves (Dobson et al., 2001). Although rabbits are much less mobile than hares, and less likely t o br each qua rantine ba rriers, t he r ole of t he r abbit c ould be conveniently as sessed during this same investigation. The aim of this thesis was to assess if and to what extent the hare and the rabbit populations were infested with internal nematode parasites of sheep in natural situations in Australia. To fulfil the research aim, the research had the following specific objectives: Analyse the gastrointestinal tracts of hares and rabbits obtained from the vicinity of sheep in three di fferent r egions of s outh-eastern A ustralia s eeking o vine n ematode p arasites an d quantify and identify nematode parasites to species through spicule morphology and confirm those findings with conventional PCR.

Thesis structure In chapter 2, M orphology, I will give a brief description of the nematode parasites found in the g astro-intestinal tr acts o f la gomorph carcasses, b ased i n av ailable k eys and s picule differentiation c haracters. S ubsequently, I w ill describe t he m ethodology ut ilized f or t his section of my work. Next, i n c hapter 3, M olecular, I w ill g ive a br ief i ntroduction t o c urrent mo lecular s tudies performed i n or der t o diagnose T richostrongylid ne matode i nfections, f ollowed b y t he description of t he m aterial a nd m ethods ut ilized t o pe rform P CR, w ith genomic D NA of nematode specimens. Chapter 4 will present the results achieved with the means provided by chapter 2 and 3. Finally, i n chapter 5, a general di scussion and c onclusion will be drawn from t he previous chapters.

20

Chapter 2

Morphology

2.1 Introduction

Traditional identification t echniques a re ba sed on m orphological e xamination of fine reproductive structures pr esent i n t he c audal e nd of i ndividual a dult m ale and f emale nematodes. However, the m ost ch aracteristic f eatures f or s pecies i dentification, in Trichostrongylids, a re t he s picules a nd t he bu rsal r ays (Nagaty, 193 2; Clapham, 1947 ; Gibbons and Khalil, 1982).

2.2 Species descriptions

2.2.1 Graphidium strigosum (Dujardin, 1845) Railliet and Henry, 1909

G. strigosum (Fig. 2.1) is t he onl y species member of t he genus Graphidium (Order Strongylida, family Trichostrongylidae, sub-family Ostertagiinae) (Massoni et al., 2011). The worms ar e bright r ed due t o t heir bl ood f eeding ha bit. T he m ale w orm i s e asily distinguishable from the female due to its well-developed caudal bursa. The male’s body is 8- 16 mm lo ng w hilst th e female is 9 -21 m m l ong (Evans, 1939 ; Durette-Desset an d Denke, 1978). S picules are 1.1 to 2.4 m m l ong (Neveu-Lemaire, 1936 ; Durette-Desset and Denke, 1978). The gubernaculum is about 93 μm long (Massoni et al., 2011).

21

Fig. 2.1 Posterior end of a male G. strigosum. Spicules and bursa are shown.

22

2.2.2 Trichostrongylus retortaeformis (Zeder, 1800)

Lagomorphs are also na tural hos ts of t he gastro-intestinal nematode T. retortaeformis (Fig. 2.2) (Order Strongylida, family Trichostrongylidae, sub-family Trichostrongylinae). The male worm is 5-7 mm long, and the female is 6-9 mm long (Neveu-Lemaire, 1936; Levine, 1968; Soulsby, 1968). There are discrepancies between reports of spicules length: 115-158 µm long (Nagaty, 1932), 100-110 µm (Neveu-Lemaire, 1936), 120-140 µm (Mönnig, 1947; Soulsby, 1968) or 129 µm long (Clapham, 1947). The two spicules of a pair are similar in size and their distal portion is bent at an acute angle. The gubernaculum is boat shaped and 40-79 µm long (Nagaty, 1932) or 63-72 µm long (Clapham, 1947).

Fig. 2.2 Posterior end of a male T. retortaeformis showing spicules, gubernaculum and bursa

23

2.2.3 Trichostrongylus colubriformis (Giles, 1892)

T. colubriformis (Fig. 2.3) (O rder Strongylida, f amily T richostrongylidae, s ub-family Trichostrongylinae) is amongst the most common nematode parasites of ruminants, especially sheep. The lengths of the male and the female worms of this species are 4-7 mm and 5-8 mm long, respectively. The spicules are long and slender, equal in size and terminate in a barb-like tip. They measure 120-171 µm. The gubernaculum has a t ypical boat shape and is 64-88 µm long (Nagaty, 1932; Ghasemikhah et al., 2011).

Fig. 2.3 Posterior end of male T. colubriformis showing spicules, gubernaculum and bursa

24

2.2.4 Trichostrongylus rugatus (Mönnig, 1925)

T. rugatus (Fig. 2.4) (O rder Strongylida, f amily T richostrongylidae, s ub-family Trichostrongylinae) is a n in testinal n ematode p arasite of r uminants. T he m ale w orm i s 4 -6 mm long and the female is 5-8 mm long. The spicules are stout, unequal in size and present characteristic creases near the caudal end. They measure 135-152 µm. The gubernaculum is 79-88 µm long (Nagaty, 1932; Neveu-Lemaire, 1936).

Fig. 2.4 Posterior end of a male T. rugatus showing spicules, gubernaculum and bursa

25

2.2.5 Passalurus ambiguus (Rudolphi, 1819)

P. ambiguus (Fig. 2.5) (Order Oxyurida, family Oxyuridae) is a stout, whitish “pinworm” that occurs in the caecum and the colon of lagomorphs worldwide (Rinaldi et al., 2007). The male is 4-5 mm long and the female is 9-11 mm long. The spicules are 90-120 µm (Soulsby, 1968).

Fig. 2.5 Male specimen of Passalurus ambiguus - posterior end is on the left upper corner

26

2.3 Methods

2.3.1 Study sites

2.3.1.1 Site selection criteria

At all sites, sheep populations were sympatric with a lagomorph population: hares, rabbits or both. Sampling sites included paddocks previously grazed by sheep or in vineyards adjacent to sheep pastures. Lagomorph carcasses were acquired from November 2010 to August 2012 from the Adelaide region of South Australia, the western district of Victoria near Hamilton (hares only) and the central western New South Wales near Wagga Wagga. Maps of sampling sites are shown in figures 2.6 and 2.7 as follows:

Fig. 2.6 Hare sampling areas in south eastern Australia. * Hare icon size is relative to sample size.

27

Fig. 2.7 Rabbit sampling areas in south eastern Australia. * Rabbit icon size is relative to sample size

28

2.3.1.2 South Australia site

The main source of hare carcasses was South Australia with 13 different sampling sites near the Adelaide region (Fig. 2.8 A-M). Most hare samples were collected in vineyards on a farm located in Langhorne Creek, 18 km from Strathalbyn. The vineyards were in the immediate vicinity of sheep pa stures. A nother s ignificant s ampling s ite w as A shbourne, 11 km f rom Strathalbyn which contributed 21 hares and six rabbits. Seven rabbits and three h ares were taken in Williamstown. Other sheep properties contributed only one to three hares. The grassland sampling areas of South Australia encompass two distinct climate zones near the Adelaide region, one, in the north, is characterized by hot dry summers and cold winters with a m ean m aximum a nnual t emperature o f 2 2.9°C a nd a m inimum of 10.7°C ( highest temperature is 45.6°C and lowest is 0°C); with a mean annual rainfall of 434.8 mm, whilst the other one, in the south, in the region of Strathalbyn, belong to the same climate zone of the sampling ar eas o f t emperate V ictoria an d N ew S outh W ales, w here w inters ar e co ld an d summers are only warm. The mean maximum annual temperature is 21.6°C and the minimum is 10.1°C (highest temperature is 45°C and lowest is -3°C); the mean annual rainfall is 480.3 mm a nd th e r elative h umidity is 5 0-68%. Wet w inters with lo w s ummer r ainfalls a re characteristic here.

2.3.1.3 Victoria site

A considerable number of hare carcasses for this study was taken from only one sampling site in Victoria, at the Department of Primary Industries (DPI), Hamilton (Fig. 2.8 N). In this area, sheep, cattle and hares were sympatric. Rabbits were only spotted near the built areas. The elevation of the site is 200 m. The mean maximum annual temperature is 18.4°C and the minimum is 7 .3°C ( highest te mperature is 4 4°C a nd lo west is -4.5°C); t he m ean a nnual rainfall is 662.9 mm and the relative humidity is 80%.

2.3.1.4 New South Wales site

The c ontribution of hare car casses w as lo w a mongst th e s ix s ampling s ites o f N ew S outh Wales (Fig. 2.8 O-T). However, the main source for rabbit carcasses was a sheep farm located south of Wagga Wagga and in Weejasper. Hares were not targeted as much by shooters, as perhaps being the property owners their focus seemed to be on the high numbers of rabbits on farms. In the New South Wales sampling sites, located between elevations of 219-390 m, the mean maximum annual temperature is 21.3°C and the minimum is 9.9°C (highest t emperature i s

29 42.5°C and lowest, -7°C); the mean annual rainfall - more uniform throughout the year than in South Australia and Victoria - is 943 mm and the relative humidity is 73%.

70

60

50

40 Number of Hares

30 Number of Rabbits

20

10

0 A B C D E F G H I J K L M N O P Q R S T

Fig. 2.8 Number of leporids acquired in each of the sampling areas in south eastern Australia

A: Langhorne Creek, SA K: Angle Vale, SA

B: Angle Vale, SA L: Ponde, SA

C: Ashbourne, SA M: Murray River, SA

D: Murray Bridge airport, SA N: DPI Hamilton, VIC

E: South of Marrabel, SA O: Dunmovin, NSW

F: Williamstown, SA P: North of Wagga Wagga, NSW

G: Reeves Plains, SA Q: Ganmain/Coolamon road, NSW

H: Edinburgh, SA R: Weejasper, NSW

I: Gawler River, SA S: Illabo, NSW

J: Kapunda, SA T: South of Wagga Wagga, NSW

30

Table 2.1 Sampling collection in each state. Hares Rabbits Total South Australia 73 15 88 Victoria 31 0 31 New South Wales 6 73 79 Total 110 88 198

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2.3.2 Animal sampling

Vehicles p atrolled s tudy sites a t n ight a nd lagomorphs w ere l ocated a nd di soriented b y spotlights, then they were shot by members of the Hunting and Conservation Branch of the Sporting S hooters’ A ssociation of A ustralia ( SSAA). C arcasses w ere t ransported f rom t he field to the laboratory and refrigerated until the next day.

2.3.2.1 Age of leporids

Age of animals was recorded in 104 hares and 35 rabbits. Estimations of hares and rabbits’ ages w ere m ade us ing S troh’s m ethod w hich i nvolves pa lpation t hrough t he s kin or di rect observation t o de termine t he de gree of t he di stal e piphyseal t hickening of t he ul na. T his method is useful for distinguishing between juveniles and adults. If the epiphyseal thickening was clearly palpable, the age of the leporid corresponded to a class from six to eight months old (Fig. 2.9 A) (Bujalska et al., 1965) , if the structure was faintly perceptible, the animal was a t a bout one year old ( Fig. 2.9 B). If no t hickening c ould be f elt, t he a nimal w as considered to be over one year old (Fig. 2.9 C). Hares and rabbits were classified into only two cat egories i n t his s tudy: j uveniles ( when an imals p resented cl early palpable ep iphyseal ulnar thickening) and adults (when thickening was only faintly perceptible or not detectable). Leverets and kittens were not targeted in this study.

Fig. 2.9 Changes in the epiphyseal thickening of the ulna in the hare (Bujalska et al., 1965).

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2.3.2.2 Gender of leporids

Sex of leporids was recorded in 109 hares and 39 rabbits.

2.3.3 Nematode sampling

The gastrointestinal tract was r emoved f rom each carcass, and the stomach, small intestine and l arge i ntestine w ere s eparated and e xamined f or gross l esions. F or e ach s ector, t he contents were washed separately and extensively with tap water, poured through wire mesh screens and finally into a jar with a final aperture of 300 µm mesh fitted into its lid to remove smaller debris, then, contents were poured into Petri dishes with one drop of parasitological iodine for observation under a dissecting microscope. Total helminth counts were performed. Adult male nematodes were separated and cut in half with a razor blade, and the posterior half was c leared w ith l actophenol f or m orphological di fferentiation under a s tereo-microscope. The anterior halves were stored in ethanol 70% for post priori molecular approaches.

2.3.3.1 Spicule identification

Male nematode specimens were identified to species using spicule morphology. Spicules were observed using a compound microscope, at 20 X and 40 X magnifications, and identified to species using published identification keys (Clapham, 1947; Soulsby, 1968; Fukumoto et al., 1980; Stankiewicz et al., 1996; Sager et al., 2010). Spicules and gubernacular lengths and widths were measured with the help of the software Analysis.

33

Chapter 3

Molecular assessment: Polymerase chain reactions

3.1 Nematode diagnostics

The accurate diagnosis of parasitic diseases is essential to epidemiologic and genetic studies of h elminth p arasites and a nthelmintic e fficacy (Grant, 1994 ). Identification of s trongylid nematodes is tr aditionally based o n mo rphological c haracteristics o f ma ture d evelopmental stages. However, means for the identification of immature stages (eggs or larvae) and some female w orms a re f requently u navailable (Soulsby, 1968 ; Berry et al., 2008). F urthermore, there was a need for improved illustrated identification keys (Lichtenfels et al., 1997).

Recently, many advances in the development of molecular techniques have been made for the identification of helminth parasites, particularly with the improvement of polymerase ch ain reaction (PCR) methods that allow exponential amplification of short DNA sequences. PCR enables rapid and reliable characterization of m any different species of n ematode parasites. Examples o f the u se o f m olecular approaches i n ve terinary pa rasitology are reviewed b y McKeand (1999), Prichard and Tait (2001), Gasser et al. (2008) and Demeler et al. (2012).

Given the success of some recent DNA-based methods in developing assays utilizing the first and second internal transcribed spacers (ITS-1 and ITS-2), and ribosomal DNA (18S, 28S) for the identification of livestock strongylid parasites species, improvement and establishment of reliable co st-effective, s ensitive a nd q uantitative a ssays a re p romising (Bott e t a l., 2009 ). They can be soon expected to be of use not only in the laboratory context but also for specific characterization o f p arasitic n ematodes i n t he f ield on a daily basis. Moreover, i nestimable data can be provided for phylogenetic studies.

34

3.2 Material and methods

3.2.1 DNA extraction

The anterior ends of males and entire female adult worms (one or, grouped, up to ten animals according to availability) were fixed and stored in 70% ethanol until DNA extraction. Then, samples w ere w ashed with d istilled w ater an d p hosphate b uffered s aline (PBS) p rior t o isolation of g enomic D NA. Genomic D NA w as i solated f rom n ematodes either using QIAamp DNA Mini Kit (Qiagen) or with a manual protocol described as follows: In a 1.5 ml centrifuge t ube, 120 µ l of T E (Tris and ED TA) buffer, 9 µ l s odium dode cyl sulfate 1 0% (SDS) and 20 µl of protein kinase 3mg/ml was added with the nematode, mixed and incubated at 55oC for two hours. Then, 150 µl of phenol chloroform was added, mixed and centrifuged for two minutes at 13,000 rpm. The clear phase was transferred to a new tube to which phenol chloroform w as a gain a dded, a nd t he tube was again centrifuged. T he clear phase w as transferred to another tube and 1 ml of 99% ethanol was added. The tube was incubated in the freezer for 15 minutes. Afterwards, it was centrifuged and the liquid was drained. 180 µ l of TE buffer was added into the tube, which was, then, vortexed and incubated at 55oC for ten minutes. 20 µ l of sodium acetate 3M was added, then, 500 µ l absolute ethanol. The solution was mixed and centrifuged for one minute. The supernatant was drained and one ml of 80% ethanol w as added. The s ample w as c entrifuged f or another m inute and t he l iquid w as drained. The tube was left to dry out at room temperature for 20 minutes. Finally, 30 µl of TE buffer w as ad ded an d t he t ube w as i ncubated a t 55 oC f or one t o t wo ho urs. S amples w ere vortexed and stored in the freezer until usage.

3.2.2 Primer design

The s econd i nternal t ranscribed s pacer ( ribosomal D NA) or ITS-2 gene s equences of T. colubriformis, T. rugatus, T. vitrinus and T. retortaeformis were retrieved from existing data on G enBank, a ccession N os: H Q844229; A B503252; A B503251; H Q389232; E F427624; EF427622; X 78066; Y 14818; A Y439027; X 78064 t o de sign generic Trichostrongylus spp. primers: 5 ’-TCGAATGGTCATTGTCAA-3’(forward); 5’ -TAAGTTTCTTTTCCTCCGCT- 3’(reverse).

35

3.2.3 Conventional PCR set up

The concentrations of MgCl2 and dNTP utilized in each final 25 µL tube were of 1.5 mM and 0.2 mM, respectively. Forward and reverse primers had a final concentration of 0.4 uM each. 1 U nit of T aq D NA pol ymerase w as ut ilized a nd 5 µL of genomic t emplate a nd ne gative controls (distilled water with no DNA) were included in the PCR runs.

PCR w as c onducted i n a Kyratec S C 2 00 t hermal cycler using t he f ollowing pa rameters: denaturation at 94oC for 45 s (38 cycles), 56oC for 45 s for annealing and 72oC for 1 min for extension. The PCR run was finalised with another final extension at 72oC for 5 min and one cooling cycle at 20oC.

Subsequently, amplicons were subjected to electrophoresis in 2% agarose gel as described by Bott (2009) and t he l engths w ere c ompared t o a 100 bp l adder ( brand). R esults of gel electrophoresis are shown in Fig. 3.1

36

Fig. 3.1 Amplicons of c. 350 bp in agarose gel, NTC = negative control

37

PCR pr oducts w ere s ubmitted f or s equencing t o t he A ustralian G enome R esearch Facility Ltd., A delaide, A ustralia. S equences w ere a ligned w ith BioEdit 7.1.3 a nd c onsensus sequences were analysed with Nucleotide BLAST®. 52 DNA sequences (internal transcribed spacer 2 ( ITS-2), c omplete s equence; a nd 28S r ibosomal R NA g ene, pa rtial s equence) of Trichostrongylus species f ound i n t his s tudy were de posited i n G enBank®. T he a ccession numbers are in table 3.1.

38

Species Host Accession number

Trichostrongylus retortaeformis Hare JX046418.1 Trichostrongylus colubriformis Hare BankIt1600253 Seq1 KC521364 Trichostrongylus colubriformis Hare BankIt1600253 Seq2 KC521365 Trichostrongylus colubriformis Hare BankIt1600253 Seq3 KC521366 Trichostrongylus colubriformis Hare BankIt1600253 Seq4 KC521367 Trichostrongylus colubriformis Hare BankIt1600253 Seq5 KC521368 Trichostrongylus colubriformis Hare BankIt1600253 Seq6 KC521369 Trichostrongylus colubriformis Hare BankIt1600253 Seq7 KC521370 Trichostrongylus retortaeformis Hare BankIt1600253 Seq8 KC521371 Trichostrongylus colubriformis Hare BankIt1600253 Seq9 KC521372 Trichostrongylus colubriformis Hare BankIt1600253 Seq10KC521373 Trichostrongylus colubriformis Hare BankIt1600253 Seq11KC521374 Trichostrongylus colubriformis Hare BankIt1600253 Seq12KC521375 Trichostrongylus retortaeformis Hare BankIt1600253 Seq13KC521376 Trichostrongylus colubriformis Hare BankIt1600253 Seq14KC521377 Trichostrongylus colubriformis Hare BankIt1600253 Seq15KC521378 Trichostrongylus colubriformis Hare BankIt1600253 Seq16KC521379 Trichostrongylus colubriformis Hare BankIt1600253 Seq17KC521380 Trichostrongylus colubriformis Hare BankIt1600253 Seq18KC521381 Trichostrongylus colubriformis Hare BankIt1600253 Seq19KC521382 Trichostrongylus colubriformis Hare BankIt1600253 Seq20KC521383 Trichostrongylus colubriformis Hare BankIt1600253 Seq21KC521384 Trichostrongylus colubriformis Hare BankIt1600253 Seq22KC521385 Trichostrongylus colubriformis Hare BankIt1600253 Seq23KC521386 Trichostrongylus colubriformis Hare BankIt1600253 Seq24KC521387 Trichostrongylus retortaeformis Hare BankIt1600253 Seq25KC521388 Trichostrongylus colubriformis Hare BankIt1600253 Seq26KC521389 Trichostrongylus colubriformis Hare BankIt1600253 Seq27KC521390 Trichostrongylus retortaeformis Hare BankIt1600253 Seq28KC521391 Trichostrongylus retortaeformis Hare BankIt1600253 Seq29KC521392 Trichostrongylus colubriformis Hare BankIt1600253 Seq30KC521393 Trichostrongylus colubriformis Hare BankIt1600253 Seq31KC521394 Trichostrongylus rugatus Hare BankIt1600253 Seq32KC521395 Trichostrongylus rugatus Hare BankIt1600253 Seq33KC521396 Trichostrongylus retortaeformis Hare BankIt1600253 Seq34KC521397 Trichostrongylus retortaeformis Hare BankIt1600253 Seq35KC521398 Trichostrongylus retortaeformis Hare BankIt1600253 Seq36KC521399 Trichostrongylus retortaeformis Rabbit BankIt1600253 Seq37KC521400 Trichostrongylus retortaeformis Rabbit BankIt1600253 Seq38KC521401 Trichostrongylus retortaeformis Rabbit BankIt1600253 Seq39KC521402 Trichostrongylus retortaeformis Rabbit BankIt1600253 Seq40KC521403 Trichostrongylus retortaeformis Rabbit BankIt1600253 Seq41KC521404 Trichostrongylus retortaeformis Rabbit BankIt1600253 Seq42KC521405 Trichostrongylus retortaeformis Rabbit BankIt1600253 Seq43KC521406 Trichostrongylus retortaeformis Rabbit BankIt1600253 Seq44KC521407 Trichostrongylus retortaeformis Rabbit BankIt1600253 Seq45KC521408 Trichostrongylus retortaeformis Rabbit BankIt1600253 Seq46KC521409 Trichostrongylus retortaeformis Rabbit BankIt1600253 Seq47KC521410 Trichostrongylus retortaeformis Rabbit BankIt1600253 Seq48KC521411 Trichostrongylus retortaeformis Rabbit BankIt1600253 Seq49KC521412 Trichostrongylus colubriformis Rabbit BankIt1600253 Seq50KC521413 Trichostrongylus retortaeformis Rabbit BankIt1600253 Seq51KC521414 Table 3.1 Genbank accession numbers of sequences from Trichostrongylid species found in wild lagomorphs in Australia.

39 Chapter 4

Results

4.1 Morphological and molecular diagnosis

In t his pa rt of t he t hesis, I pr esent t he f inal, combined, r esults w hich w ere obt ained bot h through morphological and molecular techniques as described in the previous chapters.

4.1.1 Prevalence

Sampling nights: 49 (at least); unsuccessful fieldtrips: 4. Of t he t otal 110 ha res e xamined, 63 ( 57.3 % ) pr esented one or m ore s pecies of nematode parasites. Of the total of 88 r abbits examined, 81 ( 92 %) presented one or more species of nematode p arasites. Five s pecies o f gastro-intestinal n ematodes w ere recorded p arasitising both hares and rabbits, amongst them, two were ovine: T. colubriformis and T. rugatus. The latter w as r ecovered f rom f our ha res a nd on e r abbit. This is th e f irst r eport o f natural infections of T. rugatus in wild lagomorphs. Prevalence of each worm found in lagomorphs in all three sampling regions of Australia is shown in table 4.1.

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Table 4.1 Nematode parasites of lagomorphs in Australia

x ± sd Species Prevalence (%) Maximum number worms of worms retrieved recovered/animal HARES (n = 110)

G.strigosum 1 (0.9) 1 1

T.retortaeformis 15 (13.6) 21.7 ± 47.7 184 (female adult)

T.colubriformis 36 (32.7) 19.4 ± 35.1 195 (male juvenile)

T.rugatus 4 (3.6) 7.2 ± 11.8 25 (female juvenile)

P.ambiguus 1 (0.9) 34 34

RABBITS (n = 88)

G.strigosum 44 (50) 76.8 ± 115.6 598 (female juvenile)

T.retortaeformis 54 (61.4) 21.6 ± 39.4 215 (female juvenile)

T.colubriformis 3 (3.4) 15.7 ± 11.9 24 (female juvenile)

T.rugatus 1 (1.1) 1 1

P.ambiguus 5 (5.7) 7.4 ± 5.3 15

Considering t hat s ampling w as done i n t hree di fferent s tates i n A ustralia, pr evalence of parasitism according to region is shown in Table 4.2. No significant difference was found in worm burdens in the different regions analysed (P > 0.05).

41

Table 4.2 Regional comparison of nematode prevalence in hares in Australia with mean worms recovered per animal and standard deviation.

South Australia (n=73) Victoria (n=31) New South Wales (n=6) Hares n = 110 Prevalence x ± sd Prevalence x ± sd Prevalence x ± sd (%) worms (%) worms (%) worms recovered/animal recovered/animal recovered/animal G. strigosum 1 (1.4) 1 0 0 0 0 T. colubriformis 21 (28.8) 11.9 ± 12.7 11 (35.5) 28 ± 56.8 4 (66.7) 35 ± 40.1 T. rugatus 4 (5.5) 7.2 ± 11.8 0 0 0 0 T. retortaeformis 11 (15.1) 29.1 ± 54.4 3 (9.7) 1.3 ± 0.6 1 (16.7) 1 P. ambiguus 1 (1.4) 34 0 0 0 0

42

Table 4.3 Regional comparison of nematode prevalence in rabbits in Australia with mean worms recovered per animal and standard deviation.

South Australia (n=15) New South Wales (n=73) Rabbits n = 88 x ± sd x ± sd Prevalence (%) worms Prevalence (%) worms recovered/animal recovered/animal G. strigosum 3 (20) 1.7 ± 1.1 41 (56.2) 82.3 ± 118 T. colubriformis 1 (6.7) 24 2 (2.7) 11.5 ± 13.4 T. rugatus 1 (6.7) 1 0 0 T. retortaeformis 9 (60) 29.4 ± 70.3 45 (61.6) 20 ± 30.9 P. ambiguus 4 (26.7) 5.5 ± 3.7 1 (1.4) 15

In t he h are samples, 61 % of j uveniles a nd 60 % of a dults w ere i nfected b y on e o r m ore nematode species. Amongst gender classes, 57% of females and 65% of males were found to be infected. In the rabbit samples, 61% of juveniles and 60% of adults were infected; 94% of females and 88% of males were infected. Different age and gender classes were, statistically, equally affected (P > 0.05).

Chi-Square an d Fisher Exact t ests s howed t hat, i n g eneral, n ematode p arasite i nfestations were not s ignificantly d ifferent be tween h ares a nd r abbits of di fferent r egions ( P > 0.05) . However, after performing the non-parametric sign test, significant statistical difference was found in the prevalences of T. colubriformis and T. retortaeformis between the hare and rabbit populations, in South Australia. The prevalence of T. colubriformis was distinctly higher in hares, whereas T. retortaeformis occurrence was more pronounced in rabbits than in hares in (P < 0.05). T he oc currence of ovi ne or lagomorph h elminths onl y, a nd m ixed ovine/lagomorph worms in affected hares and rabbits are presented below in Figures 4.1 and 4.2. Multiple nematode parasitic infections occurred and are shown in Figure 4.3.

43

7%

21% Ovine nematodes

Lagomorph nematodes

72% Ovine and Lagomorph

Fig.4.1 Percentage of ovine nematodes affecting parasitized hares

9%

Lagomorph nematodes

Ovine and Lagomorph

91%

Fig. 4.2 Percentage of ovine nematodes affecting parasitized rabbits

44

G. stri and T. rug

T. rug, T. ret and P. amb

T. col, T. ret and P. amb

G. stri and T. ret Rabbits Hares T. ret and P. amb

T. col and T. rug

T. col and T. ret

0 5 10 15 20 25 30

Fig. 4.3 Number of hares and rabbits with mixed nematode infections

G. stri: Graphidium strigosum

T. ret: Trichostrongylus retortaeformis

T. col: Trichostrongylus colubriformis

T. rug: Trichostrongylus rugatus

P. amb: Passalurus ambiguus

45

In or der t o f ind any r elationship be tween t he f requency o f Graphidium strigosum and frequency of Trichostrongylus retortaeformis which occurred, simultaneously, in rabbits at a site in New South Wales, Spearman’s rank-order correlation was performed. N o significant correlation was found (P > 0.05). Since lagomorph carcasses were obtained in distinct seasons of the year, the impact of the latter on the prevalence of nematode parasitism on hares and rabbits was tested. There was no significant difference in prevalence of worms in the distinct seasons of the year (P > 0.05). Morphometric data of male nematodes retrieved from hares and rabbits in Australia are given in Table 4.5.

Table 4.4 Seasonal prevalence of nematode parasitism in wild lagomorphs in Australia Hares Season Spring Summer Autumn Winter Number of animals 3 16 62 29 Number affected (%) 2 (66.7) 9 (56.3) 38 (61.3) 18 (62.1) Rabbits Number of animals - 5 30 53 Number affected (%) - 4 (80) 28 (93.3) 48 (90.5)

46

Table 4.5 Measurements of some male nematode structures with means and standard deviations

T.colubriformis T.retortaeformis T.rugatus Species (n=175) x ± sd (n=235) x ± sd (n=12)x ± sd

Spicule lengths (µm) 126 ± 6.4 121 ± 13.2 129 ± 6.8

Spicule widths (µm) 22 ± 3.1 25 ± 3 42 ± 4.3

Gubernaculum 68 ± 5 64 ± 6.6 74 ± 6.9 lengths (µm)

47 4.1.2 Necroscopic examination

Macroscopic disruptions were observed at the form of thickening of portions of the duodenal mucosa (Fig. 4.4) in a f ew hares affected by lagomorph-specific T. retortaeformis. No other visible abnormalities were detected.

Fig. 4.4 Portion of hare duodenum presenting thickening of the mucosa

48 Chapter 5

Discussion

5.1 Prevalence

Natural i nfections of l agomorphs w ith ovi ne ne matodes a re c onfirmed i n s outh e astern Australia. Trichostrongylus colubriformis and T. rugatus were found t o be pr esent i n w ild lagomorphs in the field. The two species had been established experimentally by Stott et al. (2009) and w ere amongst ot her n ematodes w hich w ere a ble t o r each a dulthood i n t he abnormal hosts. Even though Teladorsagia (Ostertagia) circumcincta and smaller numbers of Nematodirus spp. and Cooperia sp. were retrieved from hares in the experiment conducted by Stott et al. (2009), and Cooperia spp. and O. circumcincta were established, in small numbers, in experimental infections of rabbits (Wood and Hansen, 1960), these species were not found in t he f ield i n t he pr esent s tudy. T he c ommon l agomorph-specific nematode p arasite Trichostrongylus retortaeformis was pr esent a nd w as e quivalent i n p revalence i n bot h lagomorph species (Table 4.1), however, the lagomorph-specific stomach worm Graphidium strigosum had relatively high prevalence (50%) in rabbit stomachs (Table 4.1) which had the same provenance: a farm located in the central western New South Wales. A prevalence of 33% of rabbits infected with G. strigosum has been recorded in Australia in restricted areas (Mykytowycz, 1956). The nematode has not been found in semi-arid areas or in sub-tropical sites an d o nly a f ew n umbers w ere p resent at a M editerranean z one s ite (Dunsmore a nd Dudzinski, 1968). In accordance with the previous studies, the only hare which was found to be infected with G. strigosum, in the present study, was from a South Australia site, which has a Mediterranean climate. According to Broekhuizen and Kemmers (1976), hares are infected by G. strigosum only when r abbits a re p resent and t his o ccurrence i s r estricted t o m ilder areas. T his i s i n a ccordance w ith t he pr esent w ork. T he ne matode G. strigosum largely infested rabbits in a single farm located in central western New South Wales. In this region, climate co nditions a re more f avourable as r ainfall ev ents ar e m ore f requent an d p ersistent (average an nual r ainfall b etween 5 00-600 m m), e ven w hen t emperatures a re hi gh a nd evaporation is fast, contributing to the ability of nematode larval stages to survive desiccation and m igrate on pa sture (Broekhuizen a nd K emmers, 1976 ). Unfortunately, although sympatric hares were present, h are c arcasses w ere not o btained f rom t his s ame ar ea and assessment o f p resence o f G. strigosum in s ympatric h ares was n ot m ade p ossible. Nevertheless, in the Australian Capital Territory, 6% of hares sympatric with G. strigosum-

49 infested rabbits have been reported with this same parasite, with an average of 50 worms and a m aximum num ber of 250 ne matodes r etrieved f rom one i ndividual ha re (Hesterman an d Kogon, 1963).

In general, in comparison with other lagomorph populations, the hares analysed here had low worm bur dens. E quivalent ne matode pa rasite burdens i n ha res w ere pr esent i n s tudies conducted b y Bordes e t a l. (2007) and D ubinsky et a l. (2010). H owever, no ovi ne w orms were r ecorded i n ei ther of t hose s tudies. It i s pos sible, none theless, t hat i n t hose s tudies - amongst others in literature – that any ovine Trichostrongylus could have been mistaken for T. retortaeformis, a s t his ne matode i s t ypically found i n l agomorphs, a nd be cause all Trichostrongylus spp. have similar sizes and the same “hair-like” appearances. Moreover, the methodology recorded for those studies does not state detailed spicule morphology was used to distinguish between the different Trichostrongylus spp.

5.2 Nematode species distribution

Factors including seasonal availability of free living stages and their survival on pasture in different geographic c onditions (Beveridge e t a l., 1989a ) influence t he di fferent l evels of infestation of sheep by Trichostrongylids as demonstrated by Bailey et al. (2009). In 1982, Beveridge and Ford indicated t hat, i n general, i n S outh Australia, T. vitrinus was the most prevalent Trichostrongylus sp. i n s heep a nd t hat i n t erms of pr edominance, t his ne matode species seemed to be more marked in more humid areas of the state. Conversely, T. rugatus was scarce in the same areas, but abundant in the drier and hotter areas in the northern regions of the state. According to the authors, T. colubriformis had a relatively constant prevalence in sheep in South Australia. However, it was not the predominant species in any particular area. Within th e Trichostrongylus spp. of m y s tudy, T. colubriformis was the m ost p revalent species i n h ares f or al l regions ex amined. T. rugatus, a co mmon s heep p arasite considered less p athogenic t han T. vitrinus and T. colubriformis, ha d a ve ry m odest c ontribution i n lagomorphs, a nd not pr ecisely oc curring i n t he drier a reas of S outh A ustralia. D espite my conjectures b ased i n t he w ork of Beveridge and F ord ( 1982) and that T. vitrinus had been found i n r abbits i n A ustralia (Roberts, 1935 ), this s pecies which i s considered t he m ost pathogenic Trichostrongylus species t o s heep (Beveridge et a l., 1989b ) was a bsent i n t he hares and r abbits a nalysed i n m y work. Additional a battoir studies could h ave determined prevalence of T. vitrinus and other nematode species in sheep sympatric with lagomorphs. As to the oxyurid lagomorph worm Passalurus ambiguus, only six animals were affected by this parasite. Its prevalence in bot h ha res a nd r abbits w as m uch l ower t han r eported b y ot her surveys (Boag, 1985; Allan et al., 1999; Foronda et al., 2003; Ashmawy et al., 2010). 50

Distribution of various nematodes in the rabbit were studied by B oag et al. (2001). In their study, G. strigosum infections i ncreased w ith hos t a ge, w hilst T. retortaeformis and P. ambiguus infections decreased i n adult animals. In t his s tudy, a ge and s ex di d not s eem to influence n ematode p arasitism in th e h osts ( P > 0.05), n either d id d ifferent geographical localities ( Table 4.2) o r d istinct s easons w ithin A ustralia ( Table 4.4). The pow er of t he statistical te sts w as, h owever, d iminished b y the large v ariability within the cl asses and because o f o ver-dispersion of pa rasites w ithin hos t s pecies. Interestingly, t he m aximum number of w orms r etrieved i n t his w ork w as hi ghest i n j uvenile a nimals ( Table 4.1) suggesting th at th eir acquired i mmunity against h elminths was, pr esumably, not f ully developed (Dobson et al., 1990; Musongong et al., 2004; Cornell et al., 2008). Leverets and kittens, which were not included in the present study, commonly present a level of immune protection transferred via maternal milk (Boag and Garson, 1993).

Ovine nematodes seemed to have a pronounced appearance amongst free-living lagomorphs in A ustralia w ith T. colubriformis being significantly m ore p revalent in h ares (Table 4.1). Unlike the hares, the rabbits were only affected by sheep nematodes when lagomorph worms were a lso pr esent, a nd t his oc curred i n onl y t hree l eporids ( Figure 4.2). H ares w ere m ore commonly i nfected b y r uminant ne matodes t han b y l agomorph-specific nematodes ( Figure 4.1). T he hi gh m obility of ha res could be an ex planation w hy t hey w ere m ore i nfected b y ovine ne matodes t han w ere t he rabbits. N ormal levels of w orm i nfestation i n s heep, t heir grazing and hence defaecation patterns on farms (White and Hall, 1998), and the home ranges (Stott, 2003b), densities, and digesta throughput (Stott, 2008), of both hares and rabbits, show that hares would be much more likely to be exposed to infective larvae of ovine origin than of lagomorph or igin i n t he s heep z one of s outh e astern A ustralia. C onversely, r abbits ha ve a concentrated grazing pattern which would considerably increase the probability of ingesting larvae o riginating f rom rabbit f aeces. D ifferences i n immune responses between hos ts may also be co nsidered. T he h ost-parasite r elationship i s c omplex a nd de velopment of hos t immune response against nematode parasitism is influenced by a number of factors including host genotype, physiologic and genetic variations of parasite species (Hein et al., 2010). It is not yet clear if the two lagomorph species would harbour same levels of ovine nematodes if they w ere bot h e qually exposed t o i nfective l arvae on pa sture. T his w ould r equire f urther investigations. In China, more than a hundred eggs per gram of faeces of Haemonchus sp. – H. contortus has interestingly presented cross-antigenicity with G. strigosum (Cuquerella and Alunda, 2009) – retrieved from naturally-infected pikas (Wang et al., 2 009). In l aboratory rabbits, H. contortus infections were not very well established since only a few adult worms

51 and no eggs were retrieved (Hutchinson and Slocombe, 1976; Mapes and Gallie, 1977). There is still question if the hare would follow the same pattern as the rabbit or as the non-leporid lagomorph pi ka. In an y case, p ermissiveness i n t he ha re a nd i n t he r abbit i s not e qual, a s indicated b y s tudies which s how hi gher s usceptibility of ha res t o t oxoplasmosis a nd t o G. strigosum infections (Broekhuizen and Kemmers, 1976; Sedlak et al., 2000).

5.3 Evolution

In t erms of e volution, i t i s know n t hat the t hree s uper-families w ithin th e s ub-order Trichostrongylina: Trichostrongyloidea, Molineoidea and Heligmosomoidea (Durette-Desset and Chabaud, 1993) include nematode p arasites of lagomorphs that occur in the same sub- family ( or genus i n t he cas e o f Trichostrongylus) a s t hose w hich p arasitize r uminants. Durette-Desset et al . (1999) suggested t hat t he T richostrongylinae w ould ha ve adapted t o ancient ruminants from early lagomorphs, probably during the Miocene period. However, if the pr emise i s t hat a ncient l agomorphs m ight h ave pa ssed on pr imitive s trongyles t o e arly ruminants during t he M iocene, t hen, i t would be l ikely t hat l agomorphs (hares, r abbits and pikas) and ruminants (e.g. sheep, goats and even cows), shared, equivalently, trichostrongylid nematodes today. My findings show that only hares and sheep seem to have a strong natural transference o f t hese n ematode p arasites. M y w ork s uggests t hat t he t ransference o f trichostrongylids from Lagomorpha to Ruminantia would have taken place more recently than the e arly M iocene. T his i nterchange w ould h ave oc curred a fter t he r adiation of bot h the Leporids and the Bovids. Moreover, as goats appear to have the same level of permissiveness to ovine trichostrongylid nematode parasites as sheep, perhaps host transference would have occurred in the Pliocene, after the rise of the tribe Caprini which is composed of both genera Capra and Ovis (Hassanin and Douzery, 1999).

5.4 Clinical disease

It was apparent that the nematode parasites retrieved from the carcasses were not in sufficient numbers to cause overt clinical illness in the hosts. Macroscopic disruptions of the intestinal mucosa w ere onl y obs erved a s t he form of t hickened nodul es ( Figure 4.4) i n a f ew h ares affected b y T. retortaeformis. D issection o f t hese n odules r evealed m ore p resence o f T. retortaeformis. M igration o f in fective s tage o f t his n ematode ( and of G. strigosum) t o t he gastro-intestinal t issue h as b een r ecorded i n t he r abbit, as a d efence mechanism o f t his parasite, w hen c onditions a re a dverse i n t he l umen (Van K uren e t a l., 2013 ). F uture hi sto-

52 pathological s tudies w ill be us eful i n or der t o u nderstand t he pa rasite d ynamics w ithin t he host. There were no macroscopic disruptions in the hare stomach which harboured G. strigosum, presumably, be cause of t he l ow bur den – only one w orm w as r etrieved. Infected r abbits presented m oderate i nfestations of G. strigosum, how ever, t here was n o a pparent clinical disease. T his i s i n acco rdance with N ickel an d H aupt (1986) and C uquerella a nd Alunda (2009) who did not find significant alterations, even in haematological parameters; and with Broekhuizen and Kemmers (1976), who suggested that rabbits do not show signs of gastric mucosal di sruptions even w ith hi gh w orm i nfestations of t hese p arasites. Hares, on t he contrary, are less tolerant to G. strigosum infections and may display hyperaemia and oedema in the stomach wall (Broekhuizen and Kemmers, 1976).

5.5 Spicule Morphology

Initially, T. retortaeformis and T. colubriformis may l ook ve ry s imilar e ven unde r microscopy. Measurements available in literature can be misleading since lengths and widths of spicules and of gubernaculums overlap many times in both species. Overall, I found that spicules of T. colubriformis have a more slender shape than the spicules of T. retortaeformis. Moreover, t he c audal e nds of t he s picules of t he l atter a re r elatively s horter, r ounder and destitute o f s omething th at r esembles a c orona ( arrows in F igure 5.1). Nagaty (1932) describes t his co rona as a p recise “h ook-like” process o f t he s picule o f T. colubriformis, which can be best visualized from its side.

Fig. 5.1 Spicules of T. retortaeformis (left) and arrows showing corona present in T .colubriformis (right), at 20 x magnification. 53

5.6 Morphometry

Measurements t aken f rom m ale co pulatory s tructures of T. retortaeformis and T. colubriformis, in my study, are in accordance with the literature. However, T. rugatus spicular and gubernacular lengths were slightly shorter than the records found in the literature. It is conceivable that the abnormal host environment induced an atypical growth of the nematode caudal end structures (Audebert and Durette-Desset, 2007).

5.7 Molecular assay

Design o f generic p rimers u tilizing ITS-2 s equences of di stinct Trichostrongylus spp., a nd subsequent PCR to detect the different species was an efficient diagnostic tool, given that the ITS-2 sequences contain homologous characters that are preserved between species (Hoste et al., 1995; Chilton et al., 1998).

It is relevant to s ay th at in th is w ork, ma le n ematodes a s w ell a s female n ematodes w ere sequenced - the m ale ne matodes t hat w ere i dentified t hrough s picule m orphological techniques had t heir s pecies confirmed, and female worms t hat could not be di agnosed vi a morphological m ethods w ere i dentified after PCR a nd s equencing. The s equence da ta deposited i n G enbank may contribute t o f uture s tudies on p hylogenetic r elationships of parasitic nematodes.

An interesting next molecular step would be the design of a real time P CR with a melting curve analysis assay. This has been proven to be more sensitive and specific, therefore, also more accurate in the determination of the different species of nematode parasites (Gasser et al., 2008; Bott et al., 2009).

54

5.8 Permissiveness and anthelmintic resistance

Trichostrongylus axei is a “generalist” nematode parasite, i nfecting small ruminants, cattle, horses, pigs and wild ungulates (Nickel and Haupt, 1986) as well as having the capacity of freely recombinating across host species (Archie and Ezenwa, 2011). Although high genetic diversity a nd pop ulation a bundance found i n t his s pecies ha s be en associated w ith a f ast development of anthelmintic resistance (Kaplan, 2004; Wang et al., 2009), T. axei presents a lower s election f or r esistance i n c omparison w ith ot her Trichostrongylus spp.. Archie an d Ezenwa (2011) explained that T. axei is more commonly found in wildlife than in livestock and t hat when gene f low is d irected from w ildlife towards domesticated hos ts, th is mig ht decrease selection for development of resistance in the nematode species. On the other hand, T. colubriformis, frequently found i n ha res i n t he pr esent s tudy, i s m uch m ore c ommon i n sheep than in wildlife, and its rate of development of resistance is recognized to be one of the highest amongst the gastro-intestinal nematode parasites of sheep (Kaplan, 2004). Therefore, it is p ertinent to r aise th e c oncern th at h ares ma y be a s ource of resistant s trains of ovi ne nematode species transported between sheep farms.

55

5.9 Future directions

Until now, hares were an unrecognized component influencing the transmission of livestock nematode parasites to sheep and the rate of development of anthelmintic resistance between farms. W hether th e E uropean h are is a ctually tr ansmitting r esistant s trains o f r uminant nematodes to sheep or, perhaps, favouring retardation of development of drug resistance by sustaining worms in refugia, there is no doubt that its role should be examined more closely. More s tudies a nd e xperimentation s hould be u ndertaken, pe rhaps w ith t he us e of genetic markers, to clarify this issue. Furthermore, livestock nematode parasites in free-living hares might be of c onservation s ignificance s ince t hese w orms can b e as sociated w ith f actors impacting the decline of populations of wild hares in many European countries. If the hare is proven to negatively influence transmission and increase rate of development of anthelmintic resistance am ongst s heep f arms, t hen n ematode parasite co ntrol an d m anagement p rograms would, perhaps, integrate options such as hare proof fencing into their practices, along with other chemical and non-chemical strategies.

In c onclusion, t he r esults o f m y work s uggest t hat ha res m ay adversely i mpact c ontrol attempts of nematode parasites of sheep in south eastern Australia, however; more scientific knowledge i s r equired i n or der t o unde rstand t his i nteraction a nd t o r ecommend t argeted strategies (i.e. hare proof fencing or hare culling). In a broader perspective, nowadays, with the increase of interspecific interactions between wildlife, livestock and humans, transmission of di seases a mongst t hem i s not unc ommon and a good m anagement of a nimal p arasitic diseases should consider all aspects involved.

56 References

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